Compositions Comprising D-Amino Acid Peptides and Methods of Production and Use Thereof for Inhibiting Autoantibodies

ABSTRACT

Compositions are disclosed that include D-amino acid peptides and that are capable of specifically binding to at least one autoantibody against a G-protein coupled receptor. The autoantibody is produced in a patient having or being predisposed to a disease condition or disorder, and the autoantibody is capable of binding to a specific epitope of the G-protein coupled receptor. Methods of production and use of said compositions are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCESTATEMENT

This application claims benefit under 35 USC 119(e) of provisionalapplication U.S. Ser. No. 61/786,758, filed Mar. 15, 2013. The entirecontents of the above-referenced patent application are expresslyincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

BACKGROUND

A large number of patients have circulating autoantibodies directedtoward specific receptors (for example, but not by way of limitation,G-protein coupled receptors (GPCR)) which activate or partially blocktheir function and are causative of a wide variety of symptoms andabnormal bodily functions. Although specific pharmacological blockade ispresently available and may provide partial relief, these agentsgenerally block the receptor rather than just the autoantibody. Thisreceptor blockade is frequently only partially effective and/or can evenmake the symptoms worse, since they do not block the site which isdirectly targeted by the antibody within the receptor complex. It isknown that a target peptide can be used to provide short term blockadeof the autoantibody, but the very rapid inactivation of thesetarget-peptides within the body makes it difficult to use them fortreatment. Cyclic peptides (constructed with L-amino acid components)which are effective in blocking antibody function are known (see U.S.Pat. No. 8,187,605); however, these cyclic peptides involve complexsynthetic processes. Thus, alternate peptide structures which haveprolonged and effective biological activity in vivo, and which do notinterfere with necessary receptor responsivity to normal receptorstimuli, are desirable. Therefore, the presently disclosed inventiveconcepts are directed to compositions containing new peptide structuresthat overcome the disadvantages and defects of the prior art, as well asmethods of production and use thereof, along with kits containing same.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically depicts the proposed interactive effects of thyroidhormone, activating autoantibodies (AAb), and increasing age on one typeof atrial fibrillation. This represents but one of many proposedinteractions of AAb with cardiac arrhythmias alone. This concept is alsopossible for the impact of these and other GPCR-AAb targets in othertissues/organs in the body of living organisms.

FIG. 2 illustrates the peptide structures of an epitope and itscorresponding RID peptide constructed in accordance with the presentlydisclosed inventive concepts. The top peptide structure is of theangiotensin type 1 receptor (AT1R) 2^(nd) extracellular loop (ECL2)L-amino acid epitope (AFHYESQ; SEQ ID NO:2) that is the target “epitope”for the activating autoantibody directed toward the AT1R. The bottompeptide structure is of the retro-inverso D-amino acid (RID) peptide(qseyhfa; SEQ ID NO:4) that has been constructed in accordance with thepresently disclosed inventive concepts and is based on the nativeL-amino acid sequence shown in the top peptide structure. The RIDpeptide serves as a “decoy” target for the activating autoantibodiesbecause of its structural similarity to the native epitope.

FIG. 3 illustrates the in vitro effects of pre-immune (pre-imm), AT1Rpost-immune (post-imm) and AT1R post-immune serum incubated with thenative AT1R 2^(nd) ECL peptide (FIG. 3A) on activation of AT1Rtransfected CHO cells. The data are expressed as Ang II equivalentvalues. There are minimal effects from the pre-immune sera, a markedincrease in activity from the rabbit sera containing the autoantibodiesto the 2^(nd) ECL epitope, and significant inhibition of activation inthe identical rabbit sera when pre-incubated with either the nativeL-amino acid peptide (FIG. 3A, upper panel) or the RID peptide (FIG. 3B,lower panel). There is no significant difference between the effects ofthe native peptide and the RID decoy peptide.

FIG. 4A illustrates the dose related contractile effect of serum from anAT1R 2^(nd) ECL peptide immunized rabbit on an isolated rat cremasterartery. The in vitro contractile changes were expressed as % change(decrease) in diameter with the increasing dosage concentration(dilution). These effects are blocked by the selective angiotensin IIreceptor blocker (ARB) losartan.

FIG. 4B (lower panel) shows the effects of serum taken from the sameanimals as in FIG. 4A in the pre-immune, post-immune, and postimmune+RID peptide (2 mg/Kg iv) for 90 minutes. There was a significantincrease of in vitro cremaster contractility (p<0.01) in the post-immunestate compared to the pre-immune values. There was a significantdecrease in activity in the post-immune serum contractility 90 minutesafter administration of the decoy RID peptide (p<0.05). A similar effectis observed with the native L-amino acid 2^(nd) ECL peptide (data notincluded).

FIG. 5 illustrates the effect of in vitro incubation of the RID peptidewith serum containing AT1R activating autoantibodies (rabbit) oncremaster artery contractility. The post-immunized rabbit sera markedlyincreased contractility (p<0.01). After 2 hrs pre-incubation with thedecoy RID peptide, these same sera had significantly lesser contractileactivity (FIG. 5A, p<0.05). The same response was observed when theserum was pre-incubated with the L-amino acid target peptide (FIG. 5B).These data demonstrate that the enzyme-resistant RID peptide hasvirtually identical affinity for the autoantibody as has its short livedL-amino acid target peptide.

FIG. 6 illustrates the effects of the AT1R-ECL2 RID peptide on bloodpressure BP) in a rabbit model of hypertension. The AT1R-ECL2 RIDpeptide had not significant effect on BP in the pre-immune state butproduced a significant drop in systolic and diastolic BP in thepost-immune state.

FIG. 7 illustrates another embodiment of a RID peptide constructed inaccordance with the presently disclosed inventive concepts. The toppeptide structure is of the β1AR ECL2 L-amino acid epitope (RCYNDPKCCD;SEQ ID NO:26) that is the target for activating autoantibodies to theβ1AR in humans. The bottom peptide structure is of the retro-inversoD-amino acid (RID) peptide (dcckpdnycr; SEQ ID NO:27) that has beenconstructed in accordance with the presently disclosed inventiveconcepts and is based on the native L-amino acid sequence shown in thetop peptide structure. The RID peptide serves as a “decoy” target forthe activating autoantibody to the β1AR.

FIG. 8 contains a flow chart depicting the protocol of the proceduresperformed in rabbits in a study to determine the effective refractoryperiod (ERP) in atrial cells in rabbit-models with pre-immune,post-immune (β1AR-immunization), and post-immune plus β1AR-ECL2 RIDpeptide administration. The ERP inversely is one parameter of thesusceptibility of the cardiac cells to induction of tachyarrhythmias.

FIG. 9 graphically illustrates the effects of β1AR-ECL2 RID peptide andthyroxine (T4) on ERP in β1AR-immunized rabbits. *P<0.05, **P<0.01 vs.pre-immune; ^(#)P<0.05, ^(##)P<0.01 vs. post-immune; ^(&)P<0.05,^(&&)P<0.01 vs. post-immune+T4.

FIG. 10 graphically illustrates that β1AR-ECL2 RID peptide reverses theshortening of the ERP (in msec) observed in β1AR-immunized rabbits whoharbor the β1AR-AAb. ^(##)P_(<)0.01 Compared with pre-immune; **P<0.01compared with post-immune using paired t test. ERP, effective refractoryperiod; RA, right atrial.

FIG. 11 shows post-immune rabbit sera containing β1AR-AAb stimulatedcAMP production in β1AR-CHO cells. Sera taken from the same rabbits 90after IV injection of the β1AR-ECL2 RID peptide (1 mg/Kg BW)demonstrated marked reduction of the ability to stimulate cAMP.

FIG. 12 graphically illustrates an in vitro stability analysis of thenative and RID peptides in human serum at 37° C., as measured by HPLC.This Figure indicates the rapid rate of in vitro proteolysis (within 30minutes) of the AT1R-ECL2 L-amino acid peptide and the resistance of theAT1R-ECL2 RID peptide to proteolysis after two hours. In addition, 80%of the RID peptide remained after 24 hours (data not shown).

FIG. 13 graphically illustrates an in vitro stability analysis of thenative and RID peptides in synthetic gastric fluid (SGF, pepsin+HCL, pH1.2) at 37° C., as measured by HPLC. This Figure indicates the rate ofproteolysis of the AT1R-ECL2 L-amino acid peptide and the AT1R-ECL2 RIDpeptide under these conditions. A similar resistance of the AR1R-ECL2RID peptide was observed in synthetic pancreatic fluid containingtrypsin at pH 6.3 (data not shown).

FIG. 14 contains the structures of an AT1R-ECL2 RID conjugatesynthesized by inserting a PEG4 spacer in between the AT1R-ECL2 RIDpeptide and either a fluorescein molecule or a3-(3-[¹²⁵I]lodo-4-hydroxyphenyl)propionate (Bolton-Hunter reagent)molecule. This construction permits insertion of reporter molecule(s)such as an isotope of fluorescein without interfering with peptidesecondary structure-AAb interactions.

FIG. 15 illustrates another embodiment of a RID peptide constructed inaccordance with the presently disclosed inventive concepts. The toppeptide structure is of the β2AR ECL2 L-amino acid epitope (HWYRAT; SEQID NO:28) that is the target for activating autoantibodies. The bottompeptide structure is of the retro-inverso D-amino acid (RID) peptide(tarywh; SEQ ID NO:29) that has been constructed in accordance with thepresently disclosed inventive concepts and is based on the nativeL-amino acid sequence shown in the top peptide structure. The RIDpeptide serves as a “decoy” target for the activating autoantibodies.

DETAILED DESCRIPTION

Before explaining the at least one non-limiting embodiment of theinventive concepts disclosed herein in detail, it is to be understoodthat the presently disclosed inventive concepts is not limited in itsapplication to the details of examples, experiments, exemplary data,and/or methods or steps as set forth in the following description, orillustrated in the drawings. The presently disclosed inventive conceptsare capable of other embodiments or of being practiced or carried out invarious ways. Also, it is to be understood that the phraseology andterminology employed herein is for purpose of description only andshould not be regarded as limiting in any way unless specificallyindicated as such.

In the following detailed description of embodiments of the presentlydisclosed inventive concepts, numerous specific details are set forth inorder to provide a more thorough understanding of the inventiveconcepts. However, it will be apparent to one of ordinary skill in theart that the inventive concepts within the disclosure may be practicedwithout these specific details. In other instances, well-known featureshave not been described in detail to avoid unnecessarily complicatingthe present disclosure.

Unless otherwise defined herein, scientific and technical terms used inconnection with the presently disclosed inventive concepts shall havethe meanings that are commonly understood by those of ordinary skill inthe art. Further, unless otherwise required by context, singular termsshall include pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989) and Coligan et al.Current Protocols in Immunology (Current Protocols, Wiley Interscience(1994)), which are incorporated herein by reference. The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

All patents, published patent applications, and non-patent publicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which this presently disclosed inventiveconcepts pertains. All patents, published patent applications, andnon-patent publications referenced in any portion of this applicationare herein expressly incorporated by reference in their entirety to thesame extent as if each individual patent or publication was specificallyand individually indicated to be incorporated by reference.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of particular embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit, and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the inventive concepts asdefined by the appended claims.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” The term “plurality” refers to “two or more”. Throughoutthis application, the term “about” is used to indicate that a valueincludes the inherent variation of error for the device, the methodbeing employed to determine the value, or the variation that existsamong the study subjects. The use of the term “at least one” will beunderstood to include one as well as any quantity more than one,including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100,etc. The term “at least one” may extend up to 100 or 1000 or more,depending on the term to which it is attached; in addition, thequantities of 100/1000 are not to be considered limiting, as higherlimits may also produce satisfactory results. In addition, the use ofthe term “at least one of X, Y and Z” will be understood to include Xalone, Y alone, and Z alone, as well as any combination of X, Y and Z.

Throughout the specification and claims, unless the context requiresotherwise, the terms “substantially” and “about” will be understood tonot be limited to the specific terms qualified by theseadjectives/adverbs, but will be understood to indicate a value includesthe inherent variation of error for the device, the method beingemployed to determine the value and/or the variation that exists amongstudy subjects. Thus, said terms allow for minor variations and/ordeviations that do not result in a significant impact thereto. Forexample, in certain instances the term “about” is used to indicate thata value includes the inherent variation of error for the device, themethod being employed to determine the value and/or the variation thatexists among study subjects; alternatively, the term “about,” hereinwhen referring to a measurable value such as an amount, a temporalduration, and the like, is meant to encompass variations of ±20% or±10%, or ±5%, or ±1%, or ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods and asunderstood by persons having ordinary skill in the art. Similarly, theterm “substantially” may also relate to 80% or higher, such as 85% orhigher, or 90% or higher, or 95% or higher, or 99% or higher, and thelike.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, “pharmaceutically acceptable” refers to those propertiesand/or substances, which are acceptable to the patient from apharmacological/toxicological point of view including bioavailabilityand patient acceptance or to the manufacturing chemist from aphysical-chemical point of view regarding composition, formulation,stability and isolatability. The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio. The phrase “pharmaceutically-acceptable carrier” asused herein means a pharmaceutically-acceptable material, composition,or vehicle, such as a liquid or solid filler, diluent, excipient, orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient.

By “biologically active” is meant the ability to modify thephysiological system of an organism. A molecule can be biologicallyactive through its own functionalities, or may be biologically activebased on its ability to activate or inhibit molecules having their ownbiological activity.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and particularlya substantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, such as more thanabout 85%, 90%, 95%, and 99%. In one embodiment, the object species ispurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “patient” as used herein includes human and veterinarysubjects. “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including human, domestic and farm animals,nonhuman primates, and any other animal that has mammary tissue.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include, but are notlimited to, individuals already having a particular condition ordisorder as well as individuals who are at risk of acquiring aparticular condition or disorder (e.g., those needingprophylactic/preventative measures). The term “treating” refers toadministering an agent to a patient for therapeutic and/orprophylactic/preventative purposes.

A “therapeutic composition” or “pharmaceutical composition” refers to anagent that may be administered in vivo to bring about a therapeuticand/or prophylactic/preventative effect.

Administering a therapeutically effective amount or prophylacticallyeffective amount is intended to provide a therapeutic benefit in thetreatment, reduction in occurrence, prevention, or management of adisease and/or cancer. The specific amount that is therapeuticallyeffective can be readily determined by the ordinary medicalpractitioner, and can vary depending on factors known in the art, suchas the type of disease/cancer, the patient's history and age, the stageof disease/cancer, and the co-administration of other agents.

A “disorder” is any condition that would benefit from treatment with thepolypeptide. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancers include, but are notlimited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial carcinoma, salivary glandcarcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

The term “effective amount” refers to an amount of a biologically activemolecule or conjugate or derivative thereof sufficient to exhibit adetectable therapeutic effect without undue adverse side effects (suchas toxicity, irritation and allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of the inventiveconcepts. The therapeutic effect may include, for example but not by wayof limitation, inhibiting the growth of undesired tissue or malignantcells. The effective amount for a subject will depend upon the type ofsubject, the subject's size and health, the nature and severity of thecondition to be treated, the method of administration, the duration oftreatment, the nature of concurrent therapy (if any), the specificformulations employed, and the like. Thus, it is not possible to specifyan exact effective amount in advance. However, the effective amount fora given situation can be determined by one of ordinary skill in the artusing routine experimentation based on the information provided herein.

As used herein, the term “concurrent therapy” is used interchangeablywith the terms “combination therapy” and “adjunct therapy”, and will beunderstood to mean that the patient in need of treatment is treated orgiven another drug for the disease in conjunction with thepharmaceutical compositions of the presently disclosed inventiveconcepts. This concurrent therapy can be sequential therapy, where thepatient is treated first with one drug and then the other drug, or thetwo drugs can be given simultaneously.

The terms “administration” and “administering” as used herein will beunderstood to include all routes of administration known in the art,including but not limited to, oral, topical, transdermal, parenteral,subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal,intravitreal and intravenous routes, including both local and systemicapplications. In addition, the compositions of the presently disclosedinventive concepts (and/or the methods of administration of same) may bedesigned to provide delayed, controlled or sustained release usingformulation techniques which are well known in the art.

The presently disclosed inventive concepts also include a pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone of the compositions described herein in combination with apharmaceutically acceptable carrier. As used herein, a “pharmaceuticallyacceptable carrier” is a pharmaceutically acceptable solvent, suspendingagent or vehicle for delivering the compositions of the presentlydisclosed inventive concepts to the human or animal. The carrier may beliquid or solid and is selected with the planned manner ofadministration in mind. Examples of pharmaceutically acceptable carriersthat may be utilized in accordance with the presently disclosedinventive concepts include, but are not limited to, PEG, liposomes,ethanol, DMSO, aqueous buffers, oils, DPPC, lipids, otherbiologically-active molecules, vaccine-adjuvants, and combinationsthereof.

As used herein, a “therapeutically effective amount” of the inhibitor orchemotherapeutic agent of the presently disclosed inventive conceptsrefers to an amount of a compound that is effective, upon single- ormultiple-dose administration to the subject, e.g., a patient, attreating, inhibiting, mitigating, reducing, modulating, or otherwiseaffecting any of the disorders, diseases, or conditions describedelsewhere herein, for example, orthostatic hypotension, or any othercondition involving a disorder, disease or condition which involvesautoantibodies to any of the receptors described herein.

While the presently disclosed inventive concepts will now be describedin connection with certain embodiments in the following examples so thataspects thereof may be more fully understood and appreciated, it is notintended to limit the invention to these particular examples. On thecontrary, it is intended to cover all alternatives, modifications andequivalents as may be included within the scope of the presentlydisclosed inventive concepts as defined herein and in the appendedclaims. Thus, the following examples, which include particularembodiments will serve to illustrate the practice of the presentlydisclosed inventive concepts, it being understood that the particularsshown are by way of example and for purposes of illustrative discussionof particular embodiments of the presently disclosed inventive conceptsonly and are presented in the cause of providing what is believed to bethe most useful and readily understood description of compounds, methodsof use, and formulation procedures as well as of the principles andconceptual aspects of the presently disclosed inventive concepts.

Turning now to the presently disclosed inventive concepts, compositionsare described herein that include at least one D-amino acid peptide, andin certain embodiments, at least one retro-inverso D-amino acid (RID)peptide. Particular D-amino acid-containing peptides that may beutilized in accordance with the presently disclosed inventive conceptswill be defined in detail in later portions of this disclosure. Thepeptides are capable of specifically binding to autoantibodies, and inparticular, to autoantibodies produced in a patient having (orpredisposed to) a disease condition/disorder. In certain embodiments,the peptides may be capable of specifically binding to autoantibodiesthat either activate or block G-protein coupled receptors.

The presently disclosed inventive concepts include an inhibitorycomposition comprising any of the D-amino acid (D-aa) peptides describedor otherwise contemplated herein.

The presently disclosed inventive concepts also include a pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone of the peptides described or otherwise contemplated herein, incombination with a pharmaceutically acceptable carrier.

The presently disclosed inventive concepts further include a method ofdecreasing the binding of an autoantibody to a target receptor. In themethod, the autoantibody is contacted with any of the compositionsdescribed or otherwise contemplated herein, whereby the D-aminoacid-containing peptide(s) present in the composition bind to theautoantibody and prevent it from binding to its target receptor.

The presently disclosed inventive concepts further include a method ofreducing the occurrence and/or severity of a disease/disorder. In themethod, any of the compositions described or otherwise contemplatedherein is administered to a patient experiencing (or predisposed to) thedisease/disorder.

The presently disclosed inventive concepts are further directed tomethods of administering any of the compositions described or otherwisecontemplated herein for treatment in the various disorders, conditions,and diseases described herein which involve any of the G-protein coupledreceptors described herein.

The use of the L-amino acid peptide epitope to block autoantibodies hasbeen considered within the prior art; however, these naturally-occurringpeptides have not been effectively applicable because of their shortsurvival time in vivo. The use of the cyclic structure described byJahns et al. (Circulation (2005) 112 (Suppl. II:5):120) appears to beuseful; however, the synthesis requirements of this construct aresignificantly more complicated than for the construct of the presentlydisclosed inventive concepts, as an entirely different means ofprotection of the cyclic peptide structure is utilized; in addition, IVinfusion is required for administration of the peptides of Jahns et al.The synthesis of the D-amino acid-containing peptide described hereincan be adapted to a very large number of circumstances whereinautoimmune diseases have a specific identifiable target epitope; inaddition, these D-amino acid-containing peptides are amenable to oraladministration and use because of their small size and inherentprotection from proteolytic degradation by gastric and intestinalenzymes. The small size of these peptides makes them amenable topenetrating certain partially impermeable membranes in the mammalianbody, including the aforesaid gastrointestinal barrier but also notexcluding the central blood brain and the renal glomerular barrier.

In particular, the present D-amino acid-containing peptides areapplicable specifically to use in diseases of man and animal that arebased on autoantibodies that either activate or block G-protein coupledreceptors. This same use of a decoy peptide is also applicable to otherdiseases wherein other autoantibodies not necessarily “activating” innature are directed toward various cells in the body, and their specifictargeted epitope has been identified. The presently disclosed inventiveconcepts include, for example but not by way of limitation, D-aminoacid-containing peptides (including, but not limited to, RID peptides)based on the epitope structure of the Angiotensin II type 1 receptor(AT1R), the adrenergic receptors β1AR and β2AR, the muscarinicacetylcholine receptors M2R and M3R, α1 autonomic receptor (α1AR), andthe dopamine receptors D1R and D2R receptors. In certain embodiments,the D-amino acid-containing peptides are based on at least a portion ofan epitope positioned with an extracellular loop (such as, but notlimited to, ECL1 and ECL2) of these receptors. The presently disclosedinventive concepts also include target peptide(s) specific for otherGPCR-directed autoantibodies, such as but not limited to, the thyroidstimulating hormone receptor (TSHR), β3-adrenergic receptor (β3AR), anendothelin receptor(s), a thrombin receptor(s), and orphan GPCR's whosefunction has yet to be determined. Further, according to the presentlydisclosed inventive concepts, for any given epitope, a D-aminoacid-containing peptide can be constructed as described herein whichwill provide at least equivalent blocking activity for the specificautoantibody to which it is directed toward, thereby decreasing theability of the autoantibody to bind to a target receptor.

In accordance with the presently disclosed inventive concepts,non-limiting examples of construction of antibody-blocking peptides areexplained herein below. The following terminology is used: ECL1 and ECL2refer to human receptor peptide sequences of the 1st and 2ndextracellular loops of G-protein coupled receptors. TMD refers to thegenerally conserved sequences known as trans membrane domains of thereceptors located upstream and downstream of the loop sequences (whereinthe loop sequences without the TMD portion are designated as SEQ ID NOs:1 and 5-18 below). L-amino acids are shown in UPPER CASE. D-amino acidsare shown in lower case. Table 1 contains portions of the sequences ofGPCRs on which the D-amino acid-containing peptides may be based;underlined sequences refer to known functional epitopes (bindingepitope) for potential and documented receptor activity target sites forautoantibodies.

TABLE 1 GPCR SEQ ID Target Site Sequence NO: AT1R ECL2(TMD)-IHRNVFFIENTNITVCAFHYESQNSTL -(TMD) 1 β1AR ECL1 (TMD)-WGRWEYGSFFCEL-(TMD) 5 β1AR ECL2 (TMD)-HWWRAESDEARRCYNDPKCCDFVTNR -(TMD) 6 β2AR ECL1(TMD)-MKMWTFGNFWC -(TMD) 7 β2AR ECL2 (TMD)-HWYRATHQEAINCYANETCCDFFTNQ-(TMD) 8 D1 ECL2 (TMD)-HKAKPTSPSDGNATSLAETIDNCDSSLSR -(TMD) (aa's164-192) 9 D2 ECL2 (TMD)-GLNNADQNECIIANPA -(TMD) (aa's 173-188) 10 M2RECL1 (TMD)-TLYTVIGYWPLGPVVCD -(TMD) 11 M2R ECL2(TMD)-VRTVEDGECYIQFFSNAAVTFGTAI -(TMD) 12 M3R ECL1(TMD)-TTYIIMNRWALGNLACD -(TMD) 13 M3R ECL2(TMD)-KRTVPPGECFIQFLSEPTITFGTAI -(TMD) 14 α1AR ECL1 (TMD)-YWAFGRVFCNIWA-(TMD) 15 α1AR ECL2 (TMD)-GWRQPAPEDETICQINEEPGYVLFS -(TMD) 16 AT1R ECL1(TMD)-TAMEYRWPFGNYLCK -(TMD) 17 AT2R ECL2(TMD)-YFRDVRTIEYLGVNACIMAFPPEKYAQWS -(TMD) (aa's 180-208) 18

L-amino acids (“L-AA”) comprise the natural autoantibody-targeted1^(st), 2^(nd), or 3^(rd) extracellular loops (ECL) of G-protein coupledreceptors (GPCR's). Table 1 lists a representative but not exclusivelisting of such targets associated with autoantibody-activatedpathophysiological conditions. The native peptide has a target structurerepresenting the sequence:

-   -   (Upstream TMD)-kX•EPITOPE•kY-(Downstream TMD),        wherein kX represents the amino acids between the upstream TMD        and the autoantibody-binding epitope of the receptor, and        wherein kY represents the amino acids between the downstream TMD        and the autoantibody-binding epitope of the receptor. EPITOPE        refers to the defined or suspected amino acid sequence of the        receptor to which the autoantibody binds and usually comprises        4-7 amino acids. The specific binding epitope of the        kX-EPITOPE-kY sequence may be known or unknown. In a particular        embodiment the RID peptide comprises D-amino acid substitutions        of at least five of the L-amino acids of the EPITOPE portion of        the receptor, and particularly the at least five central-most        residues of the EPITOPE. For example where an EPITOPE comprises        7 L-amino acids, the RID peptide would comprise D-amino acids at        least corresponding to amino acid positions 2-6 of the EPITOPE.

In accordance with the presently disclosed inventive concepts, a peptideis constructed in a retro-inverso fashion (alternative nomenclature:retro-enantio peptide) using D-amino acids in place of the naturalL-amino acids from any of the loop sequences defined above. This peptideconstruction is based on an amino acid sequence orientation representedas:

-   -   (Downstream TMD)-yk•epitope•xk -(upstream TMD).

The RID peptide does not include the TMD structures but only amino acidsselected from the sequence “yk•epitope•xk”, wherein the amino acids arein reverse order as compared to the native sequence “kX•EPITOPE•kY”,such that the carboxy-terminal residue now appears in the place of theupstream amino-terminal residue and the amino-terminal residue nowappears in the place of the downstream carboxy-terminal residue (see,for example, FIG. 2 or 6). In this peptide structure, the side chainstructures are positioned in a manner analogous to those expressed inthe original structure, but the peptide contains (and in onenon-limiting embodiment, may consist entirely of) D-amino acids that areresistant to proteolysis in mammals, including humans.

In one embodiment, the RID peptide is derived from the full sequence ofan extracellular loop of a GPCR. For example but not by way oflimitation, the RID peptide may be derived from the AT1R ECL, whereinthe underlined portion below represents the known autoantibody targetedpeptide epitope. In this example, the RID peptide is derived from theentire known autoantibody ECL sequence comprising the target epitopewithin its broad structure.

Natural sequence: (SEQ ID NO: 1)(upstream-TMD)-IHRNVFFIENTNITVCAFHYESQNSTL- (downstream-TMD) Fullretro/inverso D-aa (RID) sequence: (SEQ ID NO: 3)ltsnqseyhfacvtintneiffvnrhi.

In another embodiment, the RID peptide is based on only the specificautoantibody-binding epitope of the GPCR. The RID peptide side groupspresent as a mirror image of its L-AA enantiomer; however, the oppositeside of this same RID peptide consequentially presents as the similarside chain as its L-AA enantiomer. In the non-limiting example of theAT1R ECL, the natural sequence and RID peptide are as follows:

Natural sequence: (SEQ ID NO: 2) AFHYESQ RID sequence: (SEQ ID NO: 4)qseyhfa.

In another embodiment, the RID peptide is constructed based on asubportion of the full RID sequence, wherein the RID peptide comprisesthe “epitope” portion as well as amino acids upstream and/or downstreamof the “epitope” portion. Using the non-limiting AT1R example above, thefull RID sequence may be Itsnqseyhfacvtintneiffvnrhi (SEQ ID NO:3),while the RID peptide may be snqseyhfacv (SEQ ID NO:19). Thenon-“epitope” portion of this embodiment of the RID peptide comprisesone or more of the D-amino acids upstream of the “epitope” and/or one ormore of the D-amino acids downstream of the “epitope.”

In certain embodiments, the presently disclosed inventive concepts aredirected to a peptide comprising a sequence of D-amino acids in aretro-inverso orientation based on a sequence comprising at least fourconsecutive residues of an L-amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1 and 5-18, wherein the at least four residuesmay be any consecutive sequence within SEQ ID NOs: 1 and 5-18; incertain embodiments, the peptide comprises at least a portion of anepitope to which an autoantibody binds to the receptor comprising thespecific SEQ ID sequence.

Peptide substitutions of the D-aa of the RID peptide may include D-aminoacids individually and independently selected from groups comprising:(1) acidic amino acids; (2) basic amino acids; (3) individually andindependently selected from the group comprising Leu, Ile, Val, Met,Trp, Tyr, and Phe; (4) individually and independently selected from thegroup comprising Ser, Thr, Ala, and Gly; (5) individually andindependently selected from the group comprising Gln and Asn.

The RID peptides of the presently disclosed inventive concepts may beconstructed to have a cyclic structure. A non-limiting example of acyclic peptide is the following RID peptide:

(SEQ ID NO: 20) cyclo-ltsnqseyhfacvtintneiffvnrhi

In another embodiment, the RID peptides involve the use of multipintechnology incorporating successive overlapping peptides (1-2 aaoverlapping) of RID sequences. Use with polyclonal subject serum orderived IgG from same with ELISA techniques will permit identificationof the specific target epitope (or multiple target epitopes) within theECL of interest. This is illustrated by identification of the targetepitope, for example but not by way of limitation, for B2AR ECL2; thus,this technology permits construction of a restricted RID peptide thatmay incorporate advantageous attributes. These attributes may include“personalized application” to a patients' autoantibody specificity.

The D-amino acid-containing peptides of the presently disclosedinventive concepts possess many advantages over the prior art, includingbut not limited to: (1) the ability to withstand all natural peptidaseswhich can act on peptides comprising L-amino acids; (2) the capabilityfor withstanding gastro-enteric exposure to natural peptidases and tolimited acid hydrolysis; and (3) the potential for oral administration.

In another embodiment of the D-amino acid-containing peptides of thepresently disclosed inventive concepts, the peptide is a mixed L-aminoacid/D-amino acid peptide which comprises a binding epitope which isconstructed of L-amino acids, while upstream and downstream portions ofthe peptide comprise one or more flanking D-amino acids on each end ofthe L-amino acid portion of the peptide; these flanking D-amino acidsserve to protect the epitope containing natural L-amino acids that islocated therebetween. For example, this embodiment of a D-aminoacid-containing peptide constructed in accordance with the presentlydisclosed inventive concepts may have the structure:

-   -   xEPITOPEx,        wherein “EPITOPE” represents the specific natural target L-amino        acids, and x represents one or more D-amino acids for blocking        peptidase activity. For example but not by way of limitation, x        may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,        17, 18, 19, 20, or more D-amino acids.

Non-limiting examples of mixed L-amino acid/D-amino acid peptidesinclude the following (wherein the upper case letters indicate anepitope formed of L-amino acids, and wherein “x” is at least one D-aminoacid):

(SEQ ID NO: 21) xAFHYESQx (SEQ ID NO: 22) xHWYRATHQEx (SEQ ID NO: 23)xYWAFGRx (SEQ ID NO: 24) xAPEDETx.In particular embodiments, the length of each D-aa sequence (i.e., each“x” in the sequences above) can vary from 1 to 20, such as from 2 to 10.In one non-limiting embodiment, the peptide has two flanking D-aminoacids. A specific non-limiting example of a mixed L-amino acid/D-aminoacid peptide based upon the AT1R target peptide is as follows:

(SEQ ID NO: 25) vcAFHYESQns.The underlined upper case letters represent the natural L-amino acid,while the lower case letters represent substituted D-amino acids.

In certain embodiments, the presently disclosed inventive concepts aredirected to a peptide comprising a sequence of L-amino acids based on asequence comprising at least four consecutive residues of an L-aminoacid sequence selected from the group consisting of SEQ ID NOs: 1 and5-18, wherein the at least four residues may be any consecutive sequencewithin SEQ ID NOs: 1 and 5-18. The peptide also contains at least oneD-amino acid flanking either end of the L-amino acid sequence of atleast four consecutive residues of at least one of SEQ ID NOS:1 and5-18. In certain embodiments, the L-amino acid portion of the peptidecomprises at least a portion of an epitope to which an autoantibodybinds to the receptor comprising the specific SEQ ID sequence.

The D-amino acid-containing peptide compositions of the presentlydisclosed inventive concepts may be administered in therapeuticallyeffective amounts. An effective amount is a dosage of the compositionsufficient to provide a therapeutically or medically desirable result oreffect in the subject to which the composition is administered. Theeffective amount will vary with the particular condition being treated,the age and physical condition of the subject being treated, theseverity of the condition, the duration of the treatment, the nature ofthe concurrent or combination therapy (if any), the specific route ofadministration, and like factors within the knowledge and expertise ofthe health practitioner. For example, in connection with methodsdirected towards treating subjects having a condition characterized byorthostatic hypotension, an effective amount would be an amountsufficient to mitigate, reduce, modulate, inhibit, or otherwiseeffectively treat the condition in the subject.

The compositions of the presently disclosed inventive concepts mayfurther contain a conjugate of any of the D-amino acid-containingpeptide compositions disclosed or otherwise contemplated hereinassociated with a labeling agent. Various methods of labeling peptidesare known in the art and may be used in accordance with the presentlydisclosed inventive concepts. Examples of labels for polypeptidesinclude, but are not limited to, the following: radioisotopes orradionuclides, fluorescent labels, chemiluminescent labels, and thelike. In some embodiments, the labeling agent may be attached to thepeptide by a spacer arm of various lengths to reduce potential sterichindrance. In addition, the terms “label”, “labeling agent,” “detectablemarker,” “detection moiety,” and “reporter molecule” are usedinterchangeably herein. These conjugates are useful in variousdiagnostic methods, as discussed in more detail in the examples.

Generally, a therapeutically effective amount will vary with thesubject's age, condition, and sex, as well as the nature and extent ofthe disease in the subject, all of which can be determined by one ofordinary skill in the art. The dosage may be adjusted by the individualphysician or veterinarian, particularly in the event of anycomplication. A therapeutically effective amount is typically, but notlimited to, an amount in a range from 0.1 μg/kg to about 2000 mg/kg, orfrom 1.0 μg/kg to about 1000 mg/kg, or from about 0.1 mg/kg to about 500mg/kg, or from about 1.0 mg/kg to about 100 mg/kg, in one or more doseadministrations daily, for one or more days. If desired, the effectivedaily dose of the active compound may be administered as two, three,four, five, six, or more sub-doses, for example, administered separatelyat appropriate intervals throughout the day, optionally, in unit dosageforms. In some embodiments, the inhibitors are administered for morethan 7 days, more than 10 days, more than 14 days, or more than 20 days.In still other embodiments, the inhibitor is administered over a periodof weeks or months. In still other embodiments, the inhibitor isdelivered on alternate days. For example, the agent may be deliveredevery two days, or every three days, or every four days, or every fivedays, or every six days, or every week, or every month.

The compounds of the presently disclosed inventive concepts may beadministered alone or in combination with the above-described drugtherapies and may be administered by a variety of administration routes.The particular mode selected will depend, of course, upon the compoundselected, the condition being treated, the severity of the condition,whether the treatment is therapeutic or prophylactic, and the dosagerequired for efficacy. The methods of the presently disclosed inventiveconcepts, generally speaking, may be practiced using any mode ofadministration that is medically acceptable, meaning any mode thatproduces effective levels of the active compounds without causingclinically unacceptable adverse effects. The administration may, forexample, be oral, intraperitoneal, intra-cavity such as rectal orvaginal, transdermal, topical, nasal, inhalation, mucosal, interdermal,or parenteral routes. The term “parenteral” includes subcutaneous,intravenous, intramuscular, or infusion. Intravenous or intramuscularroutes may not be particularly suitable for long term therapy andprophylaxis. In certain embodiments, however, it may be appropriate toadminister the compound in a continuous infusion every several days, oronce a week, or every several weeks, or once a month. Intravenous orintramuscular routes may be particularly used in emergency situations.Oral administration may be used for prophylactic treatment because ofthe convenience to the patient as well as the dosing schedule. Likewise,sustained release devices as described herein may be useful in certainembodiments for prophylactic or post-surgery treatment, for example.

Particular formulations of pharmaceutical compositions of the presentlydisclosed inventive concepts for parenteral administration include, butare not limited to, sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions, or suspensions,including saline and buffered media. Parenteral vehicles include, forexample, sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers (suchas those based on Ringer's dextrose), and the like. Preservatives andother additives may also be present such as, for example,antimicrobials, anti-oxidants, chelating compounds, inert gases, and thelike. The pharmaceutical compositions may conveniently be presented inunit dosage form and may be prepared by any of the methods well known inthe art of pharmacy.

Compositions suitable for oral administration may comprise discreteunits, such as capsules, tablets, and lozenges, each containing apredetermined amount of the inhibitor. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as syrup, anelixir, or an emulsion. In yet other embodiments, the particular vehicleis a biocompatible microparticle or implant that is suitable forimplantation into the mammalian recipient.

Other embodiments of the presently disclosed inventive concepts includepharmaceutically acceptable compositions which comprise atherapeutically-effective amount of one or more of the compoundsdescribed herein, formulated together with one or more pharmaceuticallyacceptable carriers (additives) and/or diluents. As described in detailbelow, the pharmaceutically acceptable compositions may be speciallyformulated for administration in solid or liquid form, including, butnot limited to, those adapted for the following: (1) oraladministration, for example, aqueous or non-aqueous solutions orsuspensions, tablets, e.g., those targeted for buccal, sublingual, andsystemic absorption, boluses, powders, granules, pastes for applicationto the tongue; (2) parenteral administration, for example, bysubcutaneous, intramuscular, intravenous or epidural injection as, forexample, a sterile solution or suspension, or sustained-releaseformulation; (3) topical application, for example, as a cream, ointment,or a controlled-release patch or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a cream or foam; (5)sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

Some examples of materials which can serve aspharmaceutically-acceptable carriers include, but are not limited to:(1) sugars, such as lactose, glucose, and sucrose; (2) starches, such ascorn starch and potato starch; (3) cellulose, and its derivatives, suchas sodium carboxymethyl cellulose, ethyl cellulose, and celluloseacetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

Formulations of the compositions of the presently disclosed inventiveconcepts suitable for oral administration may be, but are not limitedto, in the form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia), and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresently disclosed inventive concepts as an active ingredient. Acompound of the presently disclosed inventive concepts may also beadministered as a bolus or paste.

In solid dosage forms of the compounds of the presently disclosedinventive concepts for oral administration (capsules, tablets, pills,powders, granules and the like), the compound or compounds may be mixedwith one or more pharmaceutically-acceptable carriers, including, butnot limited to, sodium citrate or dicalcium phosphate, and/or any of thefollowing: (1) fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol, glycerol monostearate, andnon-ionic surfactants; (8) absorbents, such as kaolin and bentoniteclay; (9) lubricants, such a talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof;and (10) coloring agents. In the case of capsules, tablets, and pills,the pharmaceutical compositions may also comprise buffering agents.Solid compositions of a similar type may also be employed as fillers insoft and hard-shelled gelatin capsules using such excipients as lactoseor milk sugars, as well as high molecular weight polyethylene glycolsand the like.

Liquid dosage forms for oral administration of the compounds of thepresently disclosed inventive concepts include, but are not limited to,pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups, and elixirs. In addition to the active ingredient,the liquid dosage forms may contain inert diluents commonly used in theart, such as, for example, water or other solvents, solubilizing agents,and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, coloring, perfuming, and preservativeagents. Suspensions, in addition to the active compounds, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

EXAMPLES

Examples are provided hereinbelow. However, the presently disclosed andclaimed inventive concepts are to be understood to not be limited in itsapplication to the specific experimentation, results and laboratoryprocedures. Rather, the Examples are simply provided as one of variousembodiments and are meant to be exemplary, not exhaustive.

Example 1 Exemplary GPCR's on which D-Amino Acid-Containing Peptides canbe Based

There are various G-protein coupled receptors known to haveautoantibodies that bind thereto, wherein said binding either activatesor blocks the G-protein coupled receptor. The epitopes of the GPCR towhich the autoantibodies bind can be used as models for designingautoantibody-blocking D-amino acid-containing peptides, in accordancewith the presently disclosed inventive concepts. Non-limiting examplesof these GPCRs are listed below, along with the physiologic symptomstypical of patients having autoantibody-related conditions specific forthe receptor.

Angiotensin II Type 1 Receptor (AT1R):

Hypertension is observed in 20% of the population (60 million in theUSA).

1. Primary aldosteronism is observed in 7-8% of the hypertensivepopulation: 60% of this population has idiopathic adrenal hyperplasia(IAH), while the remaining 40% has aldosterone-producing adrenal adenoma(APA). The inventor and an Italian group have demonstrated elevatedanti-AT1R receptor autoantibodies in most patients with an APA; theinventor has also demonstrated the autoantibodies in APA and in IAH.Approximately 70% are estimated using less sensitive ELISA. Sera fromthese subjects cause constriction of the cremaster artery assay, whichis a prototypical “resistance” artery representative of those arteriesknown to contribute to increased blood pressure in mammals, and this canbe blocked by a decoy peptide.

2. “Essential” hypertension without known etiology has not beencompletely screened, and the numbers are less than reported for primaryaldosteronism; and in fact might include some of these subjects.However, these autoantibodies do not stimulate aldosterone productionequally to native Ang II. They will suppress endogenous reninproduction, however, and thus these autoantibodies may still constitutea group of significant interest.

3. “Low renin essential hypertension”—It is estimated that up to 20% ofthe hypertensive population have this characteristic constellation oflow renin and low nl aldosterone.

4. Preeclampsia is frequently associated with young mothers and firstbirths. The prevalence is approximately 5-7.5% of pregnancies worldwide.Last year there were 6 million pregnancies in the USA alone. Threestudies indicate that a large percentage of these patients withpreeclampsia develop activating autoantibodies to the AT1R, and theseautoantibodies are involved in the pathophysiology associated therewith.Blockade with an angiotensin receptor blocking (ARB) agent ends upblocking the receptor to both the autoantibody and to the native Ang IIand compromises the placental-fetal blood flow, thus being a detrimentaltreatment option. Blockade of only the autoantibody would likely notlead to such compromise.

Alpha 1 Autonomic Receptor (α1AR):

Hypertension: A low percentage in one study was observed, but this studydid not use sensitive testing. Another study demonstrated up to 40% ofscreened hypertensive subjects had such autoantibodies.

Postural Tachycardia Syndrome (POTS): Regarding the prevalence thereof,it is estimated that >500,000 patients are affected in the United Statesalone. It is frequently observed in teenage girls and to a lesser extentin older individuals. The inventor has reported that 14/14 subjects sostudied with POTS have significant levels of α1AR and β1AR activatingautoantibodies that appear to be causative of their disorder.

B2 Autonomic Receptor (β2AR) and Muscarinic M3 Receptor (M3R):

Idiopathic Orthostatic Hypotension: One in every 50 subjects isestimated to have some orthostasis (excessive drop of BP upon standing),and approximately ½ have an unknown (idiopathic) etiology. The inventorhas demonstrated that a large number of these harbor autoantibodies tothe β2AR and/or M3R. Those with associated β1AR tend to have atachycardia at rest and those with associated M2R have a restingbradycardia. All tend to have decreased homeostatic autonomicresponsiveness and demonstrate postural hypotension. Use of β-blockerstends to worsen the hypotension by blocking not only the autoantibodiesincompletely but blocking the endogenous autonomic system as well.

Atrial tachyarrhythmias: The inventor has demonstrated that a very highnumber of subjects with Graves' hyperthyroidism and associated atrialfibrillation (AF) have 1 or more autoantibodies, including β1/2AR andM2R. These are known agonists that facilitate atrial fibrillation. AF isresponsible for ⅓ of the strokes in the USA and a known associate ofdecreased cardiac function. There is evidence that 30% of Graves'subjects with AF fail to return to a normal sinus rhythm aftercorrection of their hyperthyroidism. There is evidence that a portion ofthese may be maintained in AF by the co presence of these facilitatoryautoantibodies. Since more than one autoantibody is implicated, targetedpersonalized medical suppression of the antibody without affecting thereceptor appears to be a promising approach. It is established thatorthosteric blockers for the BAR are only 60-80% effective when used forinhibition of the allosteric effects of the autoantibodies.

The inventor has also demonstrated in rabbit models that theseautoantibodies have specific targeted effects on the atrial rhythm. B2ARfacilitate development of sustained atrial tachycardias. β1ARautoantibodies facilitate development of Sinus and AV nodaltachyarrhythmias and in the presence of M2 activation facilitate AF. M2Rautoantibodies with added muscarinic activation facilitate AF andventricular tachyarrhythmias. The prevalence of these autoantibodies inAF associated with organic valvular and enlarged atria is not currentlyknown, but we suspect up to 20% will have developed autoantibodies atsome time in their course.

Idiopathic Dilative Cardiomopathy: This is probably the bestcharacterized association of autoantibodies to a particular disorder. Itis estimated that >50% of patients who have Idiopathic DilativeCardiomyopathy harbor autoantibodies to the PAR. Patients who have theseautoantibodies have the worst prognosis for sudden cardiac death (SCD).

Post viral Myocarditis: Some subjects with Post viral Myocarditisdevelop autoantibodies and these may perpetuate the damage to thepatient over a prolonged period (24). The presently disclosed peptidescan be used to inactivate these autoantibodies.

Dopamine D1 (D1R) and D2 (D2R) Receptors:

A significant number of children with Pediatric AutoimmuneNeuropsychiatric Disorders Associated with Streptococcal infections(PANDA) have obsessive-compulsive ideation, motion and/or developmentdisorders. A significant number of these subjects harbor autonomicantibodies to the Dopamine D1 and D2 receptors. These autoantibodies arestimulatory and inhibitory to several important functions in the brainby activation of their respective receptors. An example is Sydenham'schorea which is a consequence of autoantibodies to the dopamine D2R/D1Rassociated with a post streptococcal infection. The availability of adecoy that could be absorbed orally or given intravenously and wouldcross the blood brain barrier would have great potential value.

Alpha2A Autonomic Receptors (α2AR):

Raynauds' syndrome is a frequent autoimmune condition associated withtemperature sensitive focal and systemic increased vasoactivity that ispainful and leads to ulceration of digits and vascular insufficiency. Itis frequently concomitant with other autoimmune diseases. Activation ofthe α2AR has been demonstrated to lead to the vasoconstriction. We aredeveloping a RID to block these autoantibodies activating this receptorto determine if this will be a therapeutically useful approach.

Thyroid Stimulating Hormone (TSH) Receptor:

This is the most prevalent type of hyperthyroidism and 90-95% of thepatients demonstrate elevated autoantibodies that activate the TSHreceptor. If the antibody target(s) on the N terminus (alpha subunit)is/are identified, then specific medical therapy would be possible usingthe RID decoy technology.

Example 2 Background and Significance of Targeting AutoAntibodiesAgainst Cardiac GPCR's

Autoantibodies in Graves' Hyperthyroidism:

B1/2AR and M2R activation “trigger” atrial ectopy and facilitateinduction and maintenance of a “substrate” for AF. Autoantibodies (AAb)against BAR and M2R in cardiomyopathies and atrial arrhythmias wereidentified in the last 20 years. A markedly increased prevalence of AAbto BAR and M2R has been demonstrated in subjects with Graves' diseasewith AF. These observations are significant since the concurrence ofexcess thyroid hormone, age, activation of BAR and M2R, and underlyingcardiac disease facilitate development of atrial tachyarrhythmias.Furthermore, therapeutic reduction of thyroid hormone concentrationalone does not lead to return of normal sinus rhythm in 30% of thesepatients, suggesting persistence of the AAb may be factorial.

Atrial Tachyarrhythmias:

Although sinus and atrial tachycardias are more common rhythmdisturbances with thyrotoxicosis, AF is most important because of highermorbidity. The prevalence of AF in hyperthyroidism increases in stepwisefashion to greater than 20% in patients older than 70. The causation ofAF in hyperthyroidism is incompletely understood and until recently hasbeen attributed solely to excess thyroid hormone, an aging heart and/orunderlying disease. There is general awareness this is anoversimplification.

It is convenient to consider 1) “triggering” (or initiating events) suchas premature atrial contractions (PAC) or atrial tachycardia and 2)“substrate” considerations that contribute to the likelihood the ectopicbeat encounters some conduction block, spatial dispersion, anddevelopment and sustenance of abnormal circular rhythms. These changesare often related as “remodeling.” It has been demonstrated thatconditions that increase or prolong the Ca²⁺ transient in the presenceof an abbreviated action potential duration (APD) lead to early afterdepolarizations (EADs) and trigger arrhythmia through alterations inNa—Ca exchange. BAR and M2R activation from any source would lead to asimilar outcome.

Effect of Autoantibodies on GPCR Function:

This complex topic is summarized in several reviews. Orthosteric ligandsgenerally work within the internal pocket formed by the unique structureof the G-protein coupled receptors (GPCR) and rely on multipleinteractions based on the structural requirements of the ligand andreceptor. Less is known about the interactions of AAb, but their size,specificity and function targeted toward the 2nd>1st and variably the3rd extracellular loop (ECL) support an allosteric function. Bornholz etal. have summarized evidence demonstrating β1AR autoantibody activationproduces an allosteric impact on isoproterenol-induced orthostericactivation/inhibition. Their studies using a FRET labeled β1ARdemonstrated a variable impact of the autoantibodies on isoproterenolstimulation of cAMP. They found some correlation of cAMP production withthe ability of the autoantibody to inhibit concurrent internalization ofthe β1AR. This effect, however, was not universal and raises thequestion whether the internalization was directly related toautoantibody effectiveness or was an epiphenomenon. Data obtained by theinventor suggests an enhancement of both β1AR and β2AR agonist effectsof isoproterenol by patient and rabbit derived β1AR and β2AR AAb. Thereare no data demonstrating primarily blocking antibodies to β1/2AR andM2R. However the inventor has identified at least 3 AAb possessingactivating capacity when the orthosteric ligand is absent but withsignificant partial agonist effects when their respective orthostericligand is present. Additional issues are raised since GPCR may act in aheterogenic fashion. Antibody binding may well alter this interactionand either impede or facilitate receptor function under certaincircumstances.

βAR Activity in Cardiomyocytes:

βAR activation increases cAMP, activates L-type Ca channels, andincreases the movement of [Ca²⁺]_(e) to [Ca²⁺]_(i). Increased cytosolicCa²⁺ enhances a repolarizing transient outward K⁺ current, whichaccelerates repolarization in myocytes. BAR/cAMP-mediatedphosphorylation enhances the repolarizing K⁺ current, acceleratingrepolarization and shortening refractoriness, and promoting bothtriggered firing and reentry. β1/2AR/cAMP-mediated phosphorylation ofphospholamban enhances Ca²⁺ uptake and overloading in the sarcoplasmicreticulum (SR), which in turn promotes spontaneous release of SR Ca²⁺and generation of delayed (DAD) and early (EAD) afterdepolarizations andresultant triggered firing. An inward (depolarizing) current, I_(f),flowing in diastole, is augmented by phosphorylation mediated by cAMP,accelerating diastolic depolarization, and promoting automatic firing inspecialized fibers in the atria. The IKs current is also sensitive toadrenergic stimulation but may not be so important in the atrial sleevemyocardium. Thus, sustained βAR activation results in increasedautomaticity, contractility, and circumstances favoring development ofatrial tachyarrhythmias. This alteration in Ca²⁺ homeostasis enhancesautomatic firing in specialized conducting cells, facilitates conduction(including the Purkinje system) and reduces the refractory period of theAV node. This electrical remodeling appears to be a major determinant ofatrial tachyarrhythmia and AF induction and its measurement is animportant parameter of AAb activity.

Cholinergic Effects on Atrial Function:

Muscarinic agonists, acting primarily through M2R, inhibit automaticityin the SA node, slow conduction and prolong the refractory period of theAV node and Purkinje fibers, and exert negative inotropic effects onatrial and ventricular myocytes. M2R agonists activate abundant I_(KACH)channels in the atrium as well as the atrial sleeves extending into thepulmonary vein apertures. This is associated with a significantreduction in the APD and the effective refractory period (ERP) in atrialcells and increases the substrate for BAR-mediated triggering of EADformation and re-entry. Acute administration of increasing dosages ofthe M2 agonist acetylcholine paradoxically will activate sympatheticneurons via the cardiac ganglia and induce atrial tachyarrhythmias andAF in the presence of a susceptible substrate.

The importance of the autonomic nervous system (ANS) for initiation andmaintenance of AF is supported by emerging evidence from both basic andclinical studies. ANS activation facilitates EAD and triggered activityby simultaneously prolonging the intracellular calcium transient(sympathetic effect) and shortening the APD (parasympathetic effect),suggesting a synergistic effect between sympathetic and parasympatheticactivation on atrial arrhythmogenesis. Sharifov et al. infusedisoproterenol and acetylcholine into the canine sinus node artery andfound the β-agonist isoproterenol increased the likelihood and ease ofAF induction with acetylcholine compared with acetylcholine alone.Similarly, Patterson et al. showed that simultaneous injections ofacetylcholine and norepinephrine into canine pulmonary vein ganglionsled to pause-dependent induction of triggered activity and arrhythmiasarising from the pulmonary veins. These data support the concept thatcombined sympathovagal activation is profibrillatory.

Thyroid hormone on atrial function: Tri-iodothyronine (T3) has an impacton cardiac function. This active thyroid hormone increases phospholambanphosphorylation similar to but not identical to that of BAR agonists.This increases activity of the Ca²⁺-ATPase regulating SR Ca²⁺ storageand shortens the APD, increases contractility, and increases thelikelihood for development of DAD when SR overloading occurs. Theseeffects appear to include alteration of the functional properties of theNa channel and the I_(k) currents. The net result of increased thyroidactivity is an increase in atrial automaticity, shortening of the APDand refractory period, and an increased likelihood for development ofafterdepolarizations. The inventor has demonstrated that these effectsare additive to those for BAR and M2R.

Graves' hyperthyroidism exhibits signs of adrenergic overactivity,including tachycardia, glucose intolerance, excess energy utilization,and autonomic instability. Circulating catecholamines, however, aregenerally suppressed, and several autonomic functions demonstrateincreased sensitivity to β blockade. This has led to the concept of anincreased sensitivity of the βAR to compensate for the decreasedcirculating catecholamines. However, experimental induction ofhyperthyroidism using exogenous thyroid in an animal model does notreproduce βAR hypersensitivity. The present disclosure is the first toreport increased autonomic sensitivity to AAb with excess thyroidhormone (see Example 4).

The effect of age on AF: Overt hyperthyroidism and aging are associatedwith AF; the reported prevalence being 5% to 20% and higher withincreasing age. The mechanisms for this propensity for AF in agedindividuals are not clear. Atrial fibrosis and stretch increase with ageand stress of cardiac diseases and certainly may be a factor inhyperthyroidism and AF since aging is an important co-factor. Evidencealso supports the concept that age reduces extrinsic control ofintrinsic cardiac ANS activity allowing focal firing from adjacentpulmonary vein (PV) atrial tissue and predisposes the elderly to AF. Theprevalence of autoimmune disorders is bipolar for the young and aging;and is associated with such changes as might occur with generation andactivity of the AAb.

Based on historical and the inventor's own data, FIG. 1 represents acurrent formulation to explain the pathophysiology of AF in hyperthyroidstates. This demonstrates the association of excess thyroid hormone,increased age, and AAb with the adrenergic and muscarinic receptors ininitiation and maintenance of the AF. This figure is used for thespecific circumstances associated with hyperthyroidism and is notexclusive of the pro-arrhythmic effect of these same AAb in other formsof cardiac arrhythmias.

Example 3 AT1R-ECL2—RID Peptide

The coexistence of agonistic autoantibodies to β1 and β2 adrenergicreceptors (β1/2AR) and muscarinic M2 and M3 receptors (M2/3R) has beenidentified in a number of patients with orthostatic hypotension (OH).These autoantibodies also may act as an etiologic or functionalcomponent of cardiomyopathy, myocarditis, and cardiac arrhythmias.Activating autoantibodies to the angiotensin II receptor (AT1R) and theal adrenergic receptor (α1AR) have also been identified as likelyetiological agents in hypertensive disorders and in postural tachycardiasyndrome (POTS). These autoantibodies occasionally are observed in thesera of healthy subjects, but generally in a lower frequency and inlower titers. Among the effects these functionally active antibodies arepredicted to have in patients are: (a) β1/2AR activation-induced restingtachycardia and β2AR-mediated peripheral vasodilatation, (b) β1/2ARinduced or associated myocardopathy, (c) M2R activation-induced restingbradycardia and inhibition of heart rate increase despite peripheralvasodilatation, and a decreased ventricular contractility despiteadrenergic stimulation, (d) M3R activation-induced vasodilatation viaendothelial nitric oxide synthase (eNOS) activation, (e) α1AR partialantagonism leading to POTS, and/or (f) AT1R induced hypertension, and(g) pre-eclampsia related fetal and maternal risks.

A balance between these coexisting and sometimes opposing autoantibodieslikely accounts for a broad range of expression in these subjects withvariable heart rate, degrees of vasodilatation, and cardiac dysfunction.These autoantibodies, by their very nature, will induce predictablehemodynamic changes. Although the autoantibody effects may not besudden, their presence will compromise appropriate compensatorycardiovascular responses to upright posture in OH patients for example.It has also been discovered that autoantibodies to α1AR and AT1R arealso operative in hypertensive patients, in those with refractoryhypertension (“Potential relevance of alpha 1.”; PLoS ONE, November2008, e 3742-), and also in those with primary hyperaldosteronism (PA).

The large number of disease states related to the presence of theseactivating autoantibodies and lack of specific therapies for theirtreatment is a significant problem for medical research, pharmacologicalresearch and development, and patient management.

Several assays have been developed to demonstrate the presence of theseautoantibodies and to measure the wide variety of biological andphysiological responses related to their presence in human disease andanimal models of the human diseases. These include ELISA, activation ofspecific receptor-transfected cells, and vasoconstriction/vasodilatationof isolated perfused cremaster arteries. These assays permit theexamination of the presence and activity of these autoantibodies invitro as well as while circulating in animal models of human disease.

The target peptides have been identified for several of these receptorcomplexes. When a rabbit is immunized with this peptide, antibodies thatfunction identically to that observed in the human can be reliablyproduced. The inventor and others have demonstrated these autoantibodiescan be blocked with the targeted L-amino acid-based peptide.

Normal peptide structure in living cells and tissues is based entirelyon L-amino acids. Peptidases in humans fail to act on peptide structuresbased on D-amino acids. The presently disclosed inventive concepts aredirected to target peptides which (1) comprise only D-amino acids (or acombination of D- and L-amino acids) for blocking action of (a)dipeptidases from either end after infusion into the living organism and(b) endopeptidases attacking selective amino acid structures. While thissubstitution of L-amino acids with D-amino acids protects the peptidefrom degradation in the body, the structure and orientation of theaccessory side chains is changed, thereby altering the interaction ofthe novel peptides with the autoantibodies targeting that epitope. Inthe present novel peptides therefore, to preserve this interaction, themolecular sequence of the respective amino acids in the peptide has beeninverted, and (2) the amino-carboxyl linkage sequence has been reversed.

As shown in FIG. 2, the epitope of the second extracellular loop (ECL)of the AT1R receptor has been used as the basis of a retro-inversoD-amino acid (“RID”) peptide according to the presently disclosedinventive concepts. The second ECL2 has the sequence:IHRNVFFIENTNITVCAFHYESQNSTL (SEQ ID NO:1), wherein the autoantibodybinding epitope is underlined, and has the sequence AFHYESQ (SEQ IDNO:2), which is the primary target for the autoantibodies to ECL2. Theretro-inverso D-amino acid version of ECL2 has the sequence:Itsnqseyhfacvtintneiffvnrhi (SEQ ID NO: 3), wherein the D-amino acidsare represented in lower case, and wherein the retro-inverso D-aminoacid version of the binding epitope sequence is underlined and has thesequence: qseyhfa (SEQ ID NO: 4).

This format, called “retro-inverso-D” (RID), is shown in FIG. 2 tocreate a “mirror image” of the original peptide comprised of L-aminoacids and has the unique feature that the reverse side of this peptideplaces all of the accessory side chains into the identical orientationas in the original L-amino acid peptide. This side consequently isavailable for interaction with the autoantibodies and is capable ofbinding it; but with the conferred protection against proteolysis thatis not shared by the L-amino acid peptide. This peptide therefore servesas a “decoy” and inhibits the autoantibody from binding to its cellmembrane bound receptor, thereby inhibiting the agonistic orantagonistic activity of the autoantibody. This effect thus decreasesthe activity of the autoantibody, and leading to clearance of the decoypeptide-autoantibody complex by known clearance mechanisms. Potentiallythis will lead to the body developing “tolerance” to this epitope andsuppression of autoantibody production.

Data provided herein were obtained with immunized rabbit serumcontaining autoantibodies that will stimulate AT1R receptors transfectedinto CHO cells. These antibodies were equally blocked in sera taken fromimmunized rabbits that were injected with either the L-amino acidpeptide or the RID peptide (FIGS. 3A-B). These data demonstrate that theaffinity of the autoantibodies to the longer lasting RID (D-amino acid)peptide of the presently disclosed inventive concepts is equivalent tothe short-lived natural (L-amino acid) target. A bioassay forAngiotensin II-like activity using an isolated perfused cremaster arteryin vitro has also been developed. FIG. 4A uses this assay to demonstratethe contractile effect of the serum from an immunized rabbit model. FIG.4B demonstrates the impaired effect of the serum taken from a rabbittreated with the RID peptide compared to its control. The short-livednatural L-amino acid peptide showed a similar inhibitory effect 90minutes after its infusion. FIGS. 5A-B demonstrate the effect of addingeither the natural L-amino acid peptide or the RID (D-amino acid)peptide to serum in vitro for 2 hours on inhibition of theautoantibody-induced contractility in the cremaster artery.

TABLE 2 Effect of AT1R-ECL2 RID Peptide on Induced AtrialTachyarrhythmias in AT1R-Immunized Young Rabbits Atrial Pre- Post-Post-Immune + Post-Immune + Tachyarrhythmias Immune Immune RID (30 min)RID (90 min) Sustained 0/12  7/12  0/10 1/12 Non-sustained 4/12 11/1210/10 1/12

Table 2 demonstrates the effect of activating autoantibodies inimmunized rabbits (n=3) on atrial tachyarrhythmias. The rabbits wereanesthetized and a co-axial catheter is inserted via the jugular veininto the heart with the stimulus electrode in the atria sinus region.The ECG was recorded before and after stimulation with acetylcholine andbrief rapid atrial stimulation (burst pacing). Induction of transientand sustained atrial tachyarrhythmias was measured during the pre-immunestate and 6 weeks after immunization with the L-amino acid target AT1R2nd ECL peptide. The average AT1R autoantibody titer was >1:1.25 millionat this point (in vitro activity data are shown in FIGS. 3, 4, and 5).The RID peptide (2 mg/Kg BW IV) was then infused over 5 minutes, and theanimals' hearts were re-stimulated after 30 minutes and 90 minutes.There was a significant increase in both sustained and non-sustainedtachyarrhythmias in the post immune animals. These included sinus,junctional, and atrial tachycardias. There was a marked decrease insustained events at 30 and 90 minutes after RID infusion. The decreasein transient events was not observed at 30 minutes but was highlysignificant at 90 minutes.

Table 2 demonstrates the impact of autoimmunization that leads toactivation of the AT1R receptor on atrial tachyarrhythmias. There is adramatic increase in sustained and non-sustained (transient) atrialevents following atrial stimulation in the immunized animals compared totheir pre-immune state. Thirty and ninety minutes after infusion of theRID peptide, there was an almost complete suppression of the sustainedevents and a dramatic decrease in the non-sustained (transient) events.

In FIG. 6, nine rabbits were immunized with a multiple antigen peptide(MAP) containing the known AT1R-AAb epitope (AFHYESQ, SEQ ID NO:2) toinduce hypertension therein. Then the impact of 0.5% Na and lo Naintakes, along with the RID AT1R-ECL2 decoy peptide, on AAb activitywere examined. The RID peptide administered IV rapidly lowered theSystolic and Diastolic BP levels back to normal.

In addition, these animals were examined for changes in their atrialstimulation threshold using the identical protocol; an increasedsensitivity to the burst pacing and Ach was demonstrated, along with amarked increase in non-sustained and sustained atrial tachyarrhythmiasincluding AF. However, these tachyarrhythmias were abolished within 90minutes of IV administration of the AT1R-ECL2-RID peptide.

These data support the use of a decoy peptide that is effective in aspecific fashion for this AT1R receptor, in accordance with thepresently disclosed inventive concepts. These data serve as a model forRID technology applied specifically to other GPCR-autoantibodyinteractions, and in general to all other autoantibody-specific targetepitope interactions.

Example 4 β1AR-ECL2—RID Peptide

Example 3 demonstrated that the AT1R-ECL2 RID decoy peptide rapidly andeffectively blocks the hypertensive effects of AT1R-AAb in the rabbitmodel. Moreover, the AT1R-ECL2 RID peptide virtually eliminated theinduction of sustained and non-sustained atrial tachyarrhythmias in thesame model without apparent sequelae. In this Example, a similarlydramatic effect of the β1AR-ECL2 RID decoy peptide was observed.

As shown in FIG. 7, the epitope of the second extracellular loop (ECL)of the β1AR receptor was used as the basis of a retro-inverso D-aminoacid (“RID”) peptide according to the presently disclosed inventiveconcepts. The top peptide structure is of the β1AR ECL2 L-amino acidepitope (RCYNDPKCCD; SEQ ID NO:2) that is the target for activatingautoantibodies. The retro-inverse D-amino acid (RID) peptide(dcckpdnycr; SEQ ID NO:27) that has been constructed in accordance withthe presently disclosed inventive concepts is shown in the bottompeptide structure, and the RID peptide is based on the native L-aminoacid sequence shown in the top peptide structure. The RID peptide servesas a “decoy” target for the activating autoantibodies against β1AR.

The RID peptide was utilized in a study measuring the effectiverefractory period (ERP) in five New Zealand white rabbits. Theprocedures performed are outlined in the flow chart of FIG. 8. The ERPwas measured during a pre-immune state and following immunization withβ1AR ECL2 peptide to generate autoantibodies thereto, as describedpreviously (Li et al. Am J Physiol Heart Circ Physiol. (2014)306:H422-428). This was followed by administration of T4 (40 μg/kg), andan additive effect was observed on induced atrial tachyarrhythmias withthe appearance of AF in a significant percentage of the T4 treatedanimals. Of particular note was the impact of each treatment on the ERP(as shown in FIG. 9). These data confirm the additive changes inelectrical modeling induced by β1AR-AAb and T4 treatment. In addition,the ERP in the rabbits was measured following administration of theβ1AR-ECL2 RID peptide 90 minutes after T4. As shown in FIG. 9, β1ARimmunization caused a significant decrease in the ERP (P<0.05), and T4effects were additive (P<0.01). However, blockade of the β1AR-AAb withRID reversed the β1AR-Aab effects but not the effect caused by T4.

FIG. 10 illustrates the mean ERP for an atrial catheter lead. The ERPdropped significantly (p<0.01) following β1AR-ECL2 immunization over sixweeks. However, IV administration of the β1AR-ECL2 RID peptide caused adramatic rise in ERP over a 90 minute period.

FIG. 11 demonstrates the effect of the β1AR-ECL2 RID peptide onimmunized rat sera on cAMP production in β1AR-transfected cells invitro. Compared to the pre-immune sera, the anti-β1AR sera significantlyincreased cAMP production (**P<0.01), while pre-incubation with the RIDpeptide for β1AR effectively blocked the sera-induced β1AR activation ofcAMP production (^(##)P<0.01).

TABLE 3 Effect of β1AR-ECL2-RID Peptide on HR and Sustained AtrialArrhythmias in β1AR-AAb + T4 Excess Animals. Post- Pre- Post- Post-immune + T4 + EP response immune immune immune + T4 β1AR-RID Heart rate188 ± 30 201 ± 31 280 ± 56**^(##) 241 ± 35**^(##+) (beats/min)#sustained 0/20  13/20**   20/20**^(#)   6/20*⁺⁺ arrhythmias/ #inductionattempts Sustained ST 0/20  9/20*  11/20**  3/20⁺ Sustained JT 0/20 2/202/20 2/20 Sustained AT 0/20 1/20 2/20 1/20 Sustained VT 0/20 0/20 3/200/20 Sustained AF 0/20 0/20 2/20 0/20 Sustained AFL 0/20 1/20 0/20 0/20AFL, atrial flutter; AF, atrial fibrillation; AT, atrial tachycardia;JT, junctional tachycardia; ST, sinus tachycardia; VT, ventriculartachycardia. *P < 0.05, **P < 0.001 vs. pre-immune; ^(#)P < 0.05, ^(##)P< 0.001 vs. post-immune; ⁺P < 0.05, ⁺⁺P < 0.001 vs. post-immune + T4.

This Example demonstrates the powerful decoy effects of RID technologyon atrial tachyarrhythmias induced and spontaneously occurring inβ1AR-ECL2 immunized rabbits.

Example 5 B2AR-ECL2—RID Peptide

As shown in FIG. 15, the epitope of the second extracellular loop (ECL)of the B2AR receptor was used as the basis of a retro-inverso D-aminoacid (“RID”) peptide according to the presently disclosed inventiveconcepts. The top peptide structure is of the β2AR ECL2 L-amino acidepitope (HWYRAT; SEQ ID NO:28) that is the target for activatingautoantibodies. The bottom peptide structure is of the retro-inversoD-amino acid (RID) peptide (tarywh; SEQ ID NO:29) that has beenconstructed in accordance with the presently disclosed inventiveconcepts and is based on the native L-amino acid sequence shown in thetop peptide structure. The RID peptide serves as a “decoy” target forthe activating autoantibodies.

Example 6 Stability, Immunogenicity, and Further Utility of the RIDPeptides

FIGS. 12 and 13 demonstrate a stability/metabolism study of theAT1R-ECL2 RID peptide. In FIG. 12, the AT1R-ECL2 L-amino acid peptideand the AT1R-ECL2 RID peptide were each separately incubated in normalhuman serum at 37° C., and proteolysis of the peptides was measured byHPLC. As can be seen in FIG. 12, rapid proteolysis of the AT1R-ECL2L-amino acid peptide was observed, with complete loss of integrity by 45minutes. In contrast, the AT2R-ECL2 RID peptide remained intact at twohours, and 80% was observed to still be intact at 24 hours (data notshown).

In FIG. 13, the AT1R-ECL2 L-amino acid peptide and the AT1R-ECL2 RIDpeptide were each separately incubated in artificial gastric juice (SGF,pepsin+HCL, pH 1.2) at 37° C., and proteolysis of the peptides wasmeasured by HPLC. The AT1R-ECL2 RID peptide remained intact in thegastric fluid for four hours, and 60% was observed to be remaining atsix hours. As expected, the AT1R-ECL2 L-amino acid peptide was morestable in the artificial gastric juice compared to the normal humanserum; however, as can be seen in FIG. 13, the native peptide wasproteolyzed at a much faster rate than the RID peptide.

In addition, the immunogenicity of the RID peptides has also beenbriefly studied. A rabbit was immunized with the β1AR-ECL2 RID peptidebut failed to demonstrate anti-RID antibodies, as measured by ELISA(data not shown). This data demonstrates that the RID peptides are alsonon-immunogenic.

While therapeutic methods of using the RID peptides have been envisionedabove, the scope of the presently disclosed inventive concepts alsoincludes diagnostic uses of the RID peptides as well. The studies shownin FIGS. 12 and 13 were conducted at high concentrations (>250 μg/ml),and these concentrations are too high for study of absorption and use aspart of a direct assay of serum binding to autoantibodies. The AT1R-ECL2RID peptide has been iodinated successfully, but the presence of theiodine atom in this molecular structure blocked binding.

Thus, a new AT1R-RID, β1AR-RID, or generalized GPCR-ECL2-RID conjugateis synthesized in which a PEG4 spacer is inserted in between the peptideand either a fluorescein molecule or a3-(3-[¹²⁵I]lodo-4-hydroxyphenyl)proprionate (Bolton-Hunter reagent)molecule that will not interfere with AAb binding (see FIG. 14). The useof Bolton-Hunter reagent for radioiodination eliminates the possibilityof iodinating the intrinsic tyrosine, which appears to be essential forthe antibody binding. This is an indirect radioiodination method inwhich an acylating reagent,N-succinimidyl-3-(4-hydroxyphenyl)proprionate, is first radioiodinatedto obtain N-succinimidyl-3-(3-[¹²⁵I]lodo-4-hydroxyphenyl)proprionate andpurified on the HPLC. The purified N-succinimidyl-3-(3-[¹²⁵I]lodo-4-hydroxyphenyl)proprionate is then covalentlycoupled to the free —NH2 group present on the peptide. This provides twoalternative probes.

Synthesis of β1AR ECL2 RID peptide with attached fluorescein or tyrosinebegins with synthesis of B1AR-RID-PEG4 by the solid phase peptidesynthesis method manually in 0.05 mmole scale using our previouslyreported conditions (Pathuri et al. Bioconjug Chem, 23:115-124, 2012).Then, commercially available N-hydroxysuccinimide (NHS)-Fluorescein isutilized for synthesizing β1AR-RID-PEG4-Fluorescein. NHS esters reactsefficiently with the free primary amino group (—NH2) of β1AR-RID-PEG4 inpH 7-9 buffers to form a stable amide bond. Radioiodination (iodine-125or 123) will be carried out using commercially available water-solubleBolton-Hunter reagent (Biochem J 133:529-539, 1973). The peptides arepurified by HPLC.

The iodinated RID peptides can be used in various imaging methods(including but not limited to, SPECT/CT systems). This permitsexamination of the distribution of the RID-¹²³I in various organs, suchas but not limited to, brain, heart, kidney, and muscle, as well as theexamination of levels in urinary excretion. The fluorescein tagged RIDpeptides can be used for non-radioactive fluorescence quantification insimilar assays. Both iodinated and fluorescein-labelled peptides will besuccessful for both assays of sera for RID-peptide specific AAb, andthese assays will provide proportionate binding and receptor activity.They will also demonstrate gastric absorption, tissue distribution, andaccess to the CNS. The primary route in non-immune animals will be viathe kidney while hepatic/splenic clearance for those with immunecomplexes. These data can thus be used in preparing these compounds forfuture clinical applications.

Although the presently disclosed inventive concepts and the advantagesthereof have been described in detail, it should be understood thatvarious changes, substitutions and alterations can be made hereinwithout departing from the spirit and scope of the presently disclosedinventive concepts as defined in the present disclosure. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the processes, compositions of matter, means,methods and steps described in the specification. As one of ordinaryskill in the art will readily appreciate from the disclosure of thepresently disclosed inventive concepts, processes, compositions ofmatter, means, methods, or steps, presently existing or later to bedeveloped that perform substantially the same function or achievesubstantially the same result as the corresponding embodiments describedherein may be utilized according to the presently disclosed inventiveconcepts. Accordingly, the presently disclosed inventive concepts areintended to include within their scope all such processes, compositionsof matter, means, methods, or steps.

1. A composition, comprising at least one retro-inverso D-amino acid(RID) peptide capable of specifically binding to at least oneautoantibody against a G-protein coupled receptor, wherein theautoantibody is produced in a patient having or being predisposed to adisease condition or disorder, the autoantibody capable of binding to aspecific epitope of the G-protein coupled receptor, and the RID peptidecomprising D-amino acids having a sequence that is a mirror image of atleast a portion of the epitope such that the D-amino acids are in areverse order compared to the native L-amino acid sequence of theepitope.
 2. The composition of claim 1, wherein the at least oneG-protein coupled receptor is selected from the group consisting ofAT1R, AT2R, β1AR, β2AR, M2R, M3R, α1AR, D1R, D2R, TSHR, β3AR, anendothelin receptor, and a thrombin receptor.
 3. The composition ofclaim 2, wherein at least a portion of the epitope comprises at leastfour consecutive amino acids of at least one of SEQ ID NOS: 1, 5-18, and26, and wherein the RID peptide comprising a sequence of D-amino acidsin retro-inverso orientation to said at least four amino acids of atleast one of SEQ ID NOS:1, 5-18, and
 26. 4. The composition of claim 1,wherein the RID peptide comprises at least five D-amino acids.
 5. Thecomposition of claim 1, wherein the RID peptide comprises at least oneof SEQ ID NOS:3, 4, 19, 20, 27, and
 29. 6. The composition of claim 2,wherein the epitope is in an extracellular loop of the G-protein coupledreceptor.
 7. The composition of claim 1 further comprising apharmaceutically acceptable carrier.
 8. The composition of claim 7,wherein the composition is formulated for oral administration.
 9. Acomposition, comprising a mixed L-amino acid/D-amino acid (L-AA/D-aa)peptide capable of specifically binding to at least one autoantibody atleast one G-protein coupled receptor, wherein the autoantibody isproduced in a patient having or being predisposed to a disease conditionor disorder, the autoantibody capable of binding to a specific epitopeof the G-protein coupled receptor, the peptide comprising at least aportion of the epitope formed of L-amino acids and at least one D-aminoacid flanking each end of the at least a portion of the epitope.
 10. Thecomposition of claim 9, wherein the at least one G-protein coupledreceptor is selected from the group consisting of AT1R, AT2R, β1AR,β2AR, M2R, M3R, α1AR, D1R, D2R, TSHR, β3AR, an endothelin receptor, anda thrombin receptor.
 11. The composition of claim 10, wherein theepitope comprises at least four consecutive amino acids of at least oneof SEQ ID NOS:1, 5-18, and
 26. 12-18. (canceled)
 19. A method oftreating postural tachycardia syndrome (POTS) in a subject in need ofsuch treatment, comprising: administering to the subject atherapeutically-effective amount of at least one retro-inverse D-aminoacid (RID) peptide capable of specifically binding to at least oneautoantibody against an α1AR receptor, wherein the autoantibody iscapable of binding to a specific epitope of the α1AR receptor, and theRID peptide comprising D-amino acids having a sequence that is a mirrorimage of at least a portion of the specific epitope such that theD-amino acids are in a reverse order compared to the native L-amino acidsequence of the specific epitope.